Diagnostic Medical Parasitology. Lynne Shore Garcia

Fixation of Parasite Specimens and Arthropods

      Adequate fixation of parasites is important not only for diagnostic procedures but also as a means of preserving positive material for personnel and student training. There are many fixatives and preservatives available; however, only the more common ones are presented here.

      Although this chapter does not include commentary related to the histopathology laboratory, some information about formalin fixatives may be helpful. Concerns about the toxicity of formalin, particularly in the quantities used in a routine histology laboratory, have led to trials of alternative methods of tissue fixation that do not require formalin. Alcohol-based fixatives have been proposed as optimal for immunohistochemical and nucleic acid methods and may be useful for diagnostic light microscopy. Some laboratories have converted to use of an alcoholic fixative (containing 56% ethanol and 20% polyethylene glycol). Although comparative scores between the two were slightly in favor of the formalin, there were no significant differences in terms of tissue architecture, cell borders, cytoplasm, nuclear contours, chromatin texture, red blood cell membranes, and uniformity of staining. Alcohol-polyethylene glycol appears to be a satisfactory alternative to formalin in routine diagnostic surgical pathology (1). In another study, Histochoice produced a staining intensity that was comparable, and in many cases superior, to that of formalin (2).

      Formalin-fixed and paraffin-embedded tissues present particular challenges for proteomic analysis. However, most archived tissues in pathology departments and tissue banks worldwide are available in this form. Different approaches to removal of the embedding medium and protein digestion have been developed, thus releasing tryptic peptides, which are suitable for analysis by liquid chromatography-mass spectrometry. Peptide identifications made using this approach are comparable to those from matched fresh frozen tissue. Apparently, a high level of sequence coverage can be seen for proteins under study (3).

      The effect of fixation on the degradation of nuclear and mitochondrial DNA in different tissues has been examined (4). Samples of different tissues were preserved in seven fixatives for periods extending from 1 to 336 days to determine which fixatives reduce the time-dependent degradation of DNA and preserve the histologic structure. For long-term storage in combination with amplification of nuclear and mitochondrial DNA, consistent results were obtained with Carnoy’s solution and glutaraldehyde. Variable results were observed for buffered formalin. In regard to comparison of the different tissues, the quantities recovered from skeletal muscles and kidneys were larger than from other tissues. These fixative studies will become even more important as molecular methods applied to fixed tissues become more common.

      Molecular characterization of morphologic change requires precise tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses of fixed tissues, morphologic and RNA integrity in rat liver was assessed when sections were fixed in 70% neutral buffered formalin, modified Davidson’s II medium, 70% ethanol, UNIFIX, modified Carnoy’s solution, modified methacarn, Bouin’s medium, phosphate-buffered saline, or 30% sucrose. Each sample was treated with standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity (5). Modified methacarn, 70% ethanol, and modified Carnoy’s solution resulted in tissue morphology providing a reasonable alternative to formalin. Modified methacarn and UNIFIX, best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was somewhat improved. Modified methacarn, 70% ethanol, and modified Carnoy’s solution provided acceptable tissue morphology and RNA quality with both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens. In another study, the PAXgene Tissue System preserves histology similarly to formalin, but does not chemically modify RNA. RNA purified from PAXgene fixed tissues performs as well as RNA from fresh frozen tissue in real-time PCR regardless of amplicon length (6).

      PCR has also been used to identify tissue-embedded

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